Circling Training Research Articles

Long-lasting developmental effects in rat offspring after maternal exposure to acetamiprid in the drinking water during gestation.

Neonicotinoids (NNTs) are a class of insecticides proposed to be safe for pest control in urban, suburban, and agricultural applications. However, little is known about their developmental effects after repeated low-dose exposures during gestation. Here, we tested a dose considered subthreshold for maternal toxicity in rats (6 mg/kg/day) by assessing several morphological, biochemical, and neurobehavioral features in preterm fetuses and developing pups after maternal administration of the NTT acetamiprid (ACP) dissolved in the drinking water during gestational days (GD) 2-19. The exploratory evaluation included monitoring maternal body weight gain, fetal viability, body weight and sex ratio, cephalic length, neonatal body weight and sex ratio, metabolic enzymes in the placenta, maternal blood and fetal liver, and anogenital distance and surface righting response during infancy. We also used the circling training test to study the integrity of the associative-spatial-motor response in adolescence. Results showed no consistent findings indicating maternal, reproductive or developmental toxicity. However, we found ACP effects on maternal body weight gain, placental butyrylcholinesterase activity, and neurobehavioral responses, suggestive of a mild toxic action. Thus, our study showed a trend for developmental susceptibility at a dose so far considered subtoxic. Although the ACP concentration in environmental samples of surface water and groundwater has been mostly reported to be much lower than that used in our study, our results suggest that the ACP point of departure used in current guidelines aimed to prevent developmental effects may need to be verified by complementary sensitive multiple-endpoint testing in the offspring.

Activity-dependent reduction of dopamine D2 receptors during a postnatal critical period of plasticity in rat striatum is not affected by prenatal haloperidol treatment

Motor activity induced in the Circling Training test (CT) during a postnatal (PN) critical period of plasticity (PN30–37) produces a long-lasting decrease in the number of binding sites and mRNA expression levels of the dopamine D2 receptor (D2R) in rat striatum. Prenatal exposure to the antipsychotic haloperidol also decreases postnatal levels of the striatal D2R in the offspring. We examined whether such fetal exposure to haloperidol could affect the activity-dependent reduction of the D2R system during the critical period. Half of the male offspring exposed to either haloperidol (2.5 mg/kg/day), i.p.) or saline during gestational days 5–18 were subjected to the CT during the critical period, while the remaining represented CT control animals. The adult number of binding sites and mRNA expression levels of the striatal D2R at PN90 were not changed by prenatal haloperidol treatment alone. On the other hand, only pups subjected to the CT during the critical period showed decreases in both studied parameters, regardless the prenatal treatment. These findings indicated that the postnatal reduction of the striatal D2R binding induced prenatally by haloperidol does not affect long-lasting activity-dependent plastic changes on the same receptor system elicited by motor activity in an ontogenetic critical period of plasticity in rat striatum.

Developmental striatal critical period of activity-dependent plasticity is also a window of susceptibility for haloperidol induced adult motor alterations

Experience-dependent plasticity during critical periods of postnatal (PN) development shapes the adult brain anatomy and function. In rat motor system, there is a critical period of activity-dependent plasticity in the striatum (PN30-37). In this period, motor activity of running in a circular path induced in the Circling Training test (CT), elicits several plasticity changes on striatal synapses. It has been recently proposed that developmental critical periods might represent a unique pharmacological window of vulnerability to induce life-lasting behavioral modifications. In this paper we tested the hypothesis of existence of a pharmacological susceptibility to induce adult alterations on motor behavior during the striatal critical period. Due to its main action on the striatum and developmental motor behavioral effects, we applied the prototypical antipsychotic haloperidol to male rats (i.p. 0.7 or 2.5 mg/kg/day) before, during or after the period of plasticity (PN20-27, PN30-37 or PN40-47 respectively). Then, in the adulthood (PN80), we evaluated induced motor activity in the CT. The results showed that only rats exposed to the D2R blocker during the period PN30-37 increased the CT activity in comparison to control rats. Moreover, only these animals also showed an increase in the spontaneous locomotor activity at the open field test. These behavioral alterations were not accompanied by permanent striatal changes either on the number of D2R binding sites or on its mRNA expression levels. In conclusion, we have shown a pharmacological susceptibility of inducing adult motor behavior alterations by haloperidol during a natural critical period of activity-dependent plasticity (PN30-37) in rat striatum development. These results also emphasize the importance of behavioral screening for pharmacological agents to be used in developmental stages of maturation.

Permanent motor activity and learning disorders induced by exposure to phenytoin during gestation and early infancy in the rat

Experimental models and clinical data indicate that the incidence of motor and learning disorders may be increased in children of epileptic mothers taking phenytoin (PHT) during pregnancy. There is little data on the vulnerability of infants to PHT-induced long-term behavioral toxicity after gestational or early life exposure (i.e., infantile convulsion therapy). We examined the persistence of alterations in circling behavior induced by exposure to PHT during gestation, infancy, or both. Pregnant Sprague–Dawley rats were injected IP with saline (SAL) or PHT (30 mg/kg/day) during gestational days (GD) 10–18. The offspring were then administered (IP) SAL or PHT (60 mg/kg/day) during postnatal days (PD) 13–23. Afterward, Circling Training tests were performed at three time points. At PD40 and PD80, the clockwise direction of circling was reinforced. At PD150, counterclockwise circling was rewarded instead. At PD40, all PHT-treated groups demonstrated increased circling velocities compared to saline-treated controls. Higher spatial error rates for direction of circling were also observed in gestation-only and infancy-only exposures. At PD80, groups exposed during gestation had higher circling velocities than control or infancy-only exposed groups. At PD150, increases in circling velocity were apparent for the reverse learning task in groups exposed during gestation. These results indicate that early postnatal exposure to PHT may exacerbate the known long-term behavioral effects of gestational exposure.

Postnatal haloperidol eliminates the deficit in circling behavior produced by prenatal exposure to the same drug

Up to 35% of pregnant women take psychotropic drugs at least once during gestation [Austin and Mitchell, 1998]. From concurrent animal and human evidence, it has been proposed that exposure to several psychoactive medications in utero or during lactation increases the risk for permanent brain disorders . Present preventive or therapy practices applied on humans for this type of long-lasting behavioral alterations are mainly based on empirical results. Here, we test an experimental approach designed to counteract a circling performance deficit that appears in Sprague–Dawley rats at puberty on exposure to the dopaminergic blocker haloperidol (HAL) during gestation [J.L. Brusés, J.M. Azcurra, The circling training: A behavioral paradigm for functional teratology testing, in: P.M. Conn (Ed.), Paradigms for the study of behavior, Acad. Press, New York, 1993, pp. 166–179. Method Neurosci. 14]. Gestational exposure to HAL (GD 5–18, 2.5 mg/kg/day ip) induced the expected circling activity decrease in the offspring at the fifth week of life. When prenatal exposure to HAL was continued through lactation (PD5–21, 1.5 mg/kg/day ip), rats otherwise showed a control-like circling performance. No difference was yet found between lactation-only, HAL-exposed pups and saline (SAL)-treated controls ( n=8 each group). We further performed saturating ( 3H)-spiroperidol (SPI) binding assays on striatal P2 membrane fractions 2 months later. The dopamine-type D2-specific binding results suggested that above circling behavior findings could be partially explained by enduring HAL-induced neurochemical changes. The role of critical periods of sensitivity as transient windows for opportunistic therapies for behavioral teratology is discussed.

Different dopamine D1 and D2 receptors expression after motor activity in the striatal critical period

Circling training during rat striatum postnatal critical period (PN30 to 37 days) induces a life-lasting co-reduction of muscarinic acetylcholine receptors (mAChR) and dopamine D2 receptors (D2R) binding. Here, we evaluated the expression of D1R and D2R under similar experimental conditions. Trained rats showed a decrease of 40% in D2R binding sites ( p<0.01) and of 45% in the D2R mRNA expression which involve short ( p<0.05) and long ( p<0.01) isoforms. In contrast, D1R binding sites nor its mRNA expression levels were affected by training, indicating a differential synaptic refinement during this ontogenetically fixed period.

Delayed and bilateral changes of GAP-43/B-50 phosphorylation after circling training during a critical period in rat striatum

During the critical period of activity-dependent plasticity in rat striatum (30–37 days after birth) physiological circling behavior induces delayed modifications in GAP-43/B-50 phosphorylation by PKC. Postexercise, ipsi- and contralateral striatum to the circling direction show a similar temporal pattern of GAP-43/B-50 phosphorylation, with an initial decrease followed by a subsequent increase. However, there is a lag between initiation of the phosphorylation response in this asymmetrical task which does not occur when animals are subjected to exercise under conditions of symmetrical motor activity.

Decreased GAP-43/B-50 phosphorylation in striatal synaptic plasma membranes after circling motor behavior during development

We evaluated the in vitro phosphorylation of the presynaptic substrate of protein kinase C (PKC), GAP-43/B-50 and the PKC activity in the striatum of rats submitted to a circling training (CT) test during postnatal development. Motor activity at 30 days of age, but not at other ages, produced a unilateral reduction (−29.5%; p<0.001) in the level of GAP-43/B-50 endogenous phosphorylation in the contralateral striatum with respect to the ipsilateral side, while non-trained control animals did not show asymmetric differences. Compared to controls, the contralateral striatum of trained animals also showed a significant reduction (−29.3%; p<0.001) in the incorporation of 32 P -phosphate into GAP-43. This decreased in vitro GAP-43 phosphorylation was seen at 30 min, but not immediately after circling motor behavior. This contralateral change in GAP-43 phosphorylation correlated with the running speed developed by the animals [( r=0.9443, p=0.0046, n=6, relative to control group) and ( r=0.8813, p=0.0203, n=6, with respect to the ipsilateral side of the exercised animals)]. On the contrary, GAP-43/B-50 immunoblots did not show changes in the amount of this phosphoprotein among the different experimental groups. Back phosphorylation assays, performed in the presence of bovine purified PKC, increased the level of GAP-43/B-50 phosphorylation in the striatum contralateral to the sense of turning [(+22%; p<0.05, with respect to ipsilateral side of the same trained group) and (+21%; p<0.05, relative to control group)]. Taken together, these results demonstrate that the activity developed in the CT test induces a reduction in the phosphorylation state of GAP-43/B-50 in the specific site for PKC. We conclude that general markers of activity-dependent neuronal plasticity are also altered in the same period that long-lasting changes in striatal neuroreceptors are triggered by circling motor behavior.

Nerve growth factor preserves a critical motor period in rat striatum.

We previously found the occurrence of a critical motor period during rat postnatal development where circling training starting the 7-day schedule at 30 days-but not before or after-induces a lifetime drop in the binding to cholinergic muscarinic receptors (mAChRs) in striatum. Here, we studied whether nerve growth factor (NGF) participates in this restricted period of muscarinic sensitivity. For this purpose, we administered mouse salival gland 2.5S NGF (1.4 or 0.4 microg/day, infused by means of ALZA minipumps) by intrastriatal unilateral route between days 25 and 39, and then trained rats starting at 40 days. Under these conditions, NGF induced a long-term reduction in the striatal [3H] quinuclidilbenzylate (QNB) binding sites despite the fact that motor training was carried out beyond the natural critical period. Thus, at day 70, measurement of specific QNB binding in infused striata of trained rats showed decreases of 42% (p < .0004) and 33% (p < .02) after administration of the higher and lower NGF doses, respectively, with respect to trained rats treated with cytochrome C, for control. Noncannulated striata of the NGF-treated rats also showed a decrease in QNB binding sites (44%; p < .0001) only at the higher infusion rate. This effect was not found in the respective control groups. Our observations show that NGF modulates the critical period in which activity-dependent mAChR setting takes place during rat striatal maturation.

Co-alteration of dopamine D2 receptor and muscarinic acetylcholine receptor binding in rat striatum after circling training.

CIRCLING training (CT) decreases muscarinic acetyl-choline receptor (mAchR) binding in rat striatum. As cholinergic and dopaminergic systems interact strongly we evaluated the expression of D2-subtype dopamine receptor (DA D2) and mAchR together after CT. Animals trained from 30 to 37 days of age and sacrificed 2 months later showed an enduring drop in Bmax of 40% in DA D2 and 34% in mAchR. Plotting the percentage of binding drop of both receptors for each animal showed that the reduction of one system correlates with the other (r2 = 0.71, p < 0.01; n = 8). Neither mAchR nor DA D2 were affected when training started at 20 or 60 days. We conclude that the presence of a period where CT exerts long term alterations during development involves both cholinergic and dopaminergic systems.

Permanent alteration of muscarinic acetylcholine receptor binding in rat striatum after circling training during development

We evaluated the effect of circling training (CT) in the expression of muscarinic acetylcholine receptor (mAchR) in developing rat striatum. For this, male and female rats were subjected to CT at 20, 30, 40 and 60 days of age during 7 days. Animals trained at 30 days but not at other ages showed an average decreased binding to mAchR of 33% in males and 24% in females, representing a significant difference with respect to control non-trained animals (males P < 0.001, females P < 0.005), and showing also a differential response between sex ( P < 0.01). mAchR drop was found invariably either 2 months or 1 year after training indicating a long term plastic change due to circling training. Scatchard analysis showed that altered binding represents a variation of the total receptor number instead of its binding affinity, with no significant differences found among K d ( P > 0.1). mAchR variation was correlated with the motor performance accomplished in the test. Regarding total distance run, male rats trained for 3 days (300 meters run), for 5 days (600 meters) and for 7 days (900 meters) showed a drop of 19, 28 and 33% respectively ( r 2 = 0.91, P < 0.001), while female changes were of 21, 23 and 24% ( r 2 = 0.78, P < 0.001). Nevertheless, no correlation with running speed was found ( r 2 = 0.13 male, r 2 = 0.02 female; P > 0.1). In summary, these results demonstrate the presence of a limited sensitivity period during striatum development where mAchR expression may be affected by the activity performed during CT, representing a permanent alteration of the receptor levels.

Preoperative operant circling training facilitates recovery following unilateral substantia nigra lesion in rats

This study was undertaken to examine (1) whether pre- and/or postoperative training, using water reinforcement, to turn in circles (rotation) affects the behavioral symptoms induced by unilateral 6-hydroxydopamine (6-OHDA)-induced DA denervation of the rat substantia nigra (SN); (2) whether there was any influence of this training on the temporal pattern of recovery; and (3) whether the rotational training influences turning induced by systemic injection of dopaminergic drugs. In the first experiment, rats were trained either ipsi- or contraversive (TI or TO) to the side to be damaged 11 days before and 23 days after lesion, and tested in an open field for rotational behavior following systemic administration of apomorphine and amphetamine. In the second experiment rats were trained only before the lesion was made and tested in the open field for spontaneous circling and thigmotactic behavior. The results of the first experiment indicated maintenance of the training performance after the lesion. At the 14th day after the lesion, the ipsiversive trained group showed a higher contraversive circling frequency after apomorphine injection in relation to the contralateral trained group. In the second experiment, rats trained only before the surgery, showed asymmetrical spontaneous circling in the trained direction before and 14 days after surgery, indicating, in a drug free condition, that training direction can be restored after unilateral SN lesions, even to the contralateral body side. Moreover, thigmotactic behavior indicated a lack of habituation in an open field in unilateral lesion rats.(ABSTRACT TRUNCATED AT 250 WORDS)

The circling training rat model as a behavioral teratology test

The properties of circling training (CT) for detecting behavioral teratologic drug-induced effects was evaluated by prenatal administration of two behavioral teratogenic drugs: vitamin A (80,000 IU/kg/day) and haloperidol (2.5 mg/kg/day). The circling training was started at 30 days of age and performed for 8 days in an automated apparatus. Statistically significant differences between drug-treated and control animals regarding the measured response (turns per minute) were found. Two components may affect the response measured by the CT: associative learning and motor performance. The incidence of these components was discriminated with behavioral and mathematical approaches. In the experimental conditions used the most affected parameter was motor performance. The results indicate that CT can be used as an instrumental conditioning test where the quantifiable endpoint is the on-going motor performance. Further applications of the CT for neurochemical evaluation of drug induced effects are also discussed.

Reversal of amphetamine-induced circling preference in trained circling rats

Others have shown that amphetamine given to normal rats causes turning in a particular, preferred direction in most animals. We have studied the effect of training on amphetamine-induced behavioral and biochemical asymmetries in male Sprague-Dawley rats. Water deprived animals were trained to circle either in the same or opposite direction to their intrinsic bias using a sucrose water reward. Acquisition of the learned circling behavior was independent of turning preference and all animals were able to make the operant association. After training, animals given amphetamine turned in the trained direction regardless of their previous circling preference. Amphetamine-induced circling also led to increased dopamine concentrations in caudate contralateral to the trained circling direction. Therefore, intrinsic striatal is not resistant to behavioral modification and both the behavioral and biochemical asymmetries can be resersed by circling training.